WO2020076914A1 - Procédé de réaction en chaîne par polymérase numérique permettant la détection d'acides nucléiques dans des échantillons - Google Patents

Procédé de réaction en chaîne par polymérase numérique permettant la détection d'acides nucléiques dans des échantillons Download PDF

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WO2020076914A1
WO2020076914A1 PCT/US2019/055342 US2019055342W WO2020076914A1 WO 2020076914 A1 WO2020076914 A1 WO 2020076914A1 US 2019055342 W US2019055342 W US 2019055342W WO 2020076914 A1 WO2020076914 A1 WO 2020076914A1
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fragment
adapter
pcr
sample
nucleic acid
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PCT/US2019/055342
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WO2020076914A9 (fr
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Kuo-Ping Chiu
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Academia Sinica
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Priority to CN201980066792.3A priority Critical patent/CN113728114A/zh
Priority to EP19871402.4A priority patent/EP3864658A4/fr
Priority to US17/283,809 priority patent/US20210340611A1/en
Publication of WO2020076914A1 publication Critical patent/WO2020076914A1/fr
Publication of WO2020076914A9 publication Critical patent/WO2020076914A9/fr

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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/20—Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/10—Signal processing, e.g. from mass spectrometry [MS] or from PCR

Definitions

  • the present invention relates to a method for detecting nucleic acid (NA) molecules in samples. More particularly, the present invention relates to an improved digital PCR-based method for analyzing nucleic acid fragments. The present invention is useful for research and diagnostic applications with increased sensitivity and accuracy. The present invention also provides a kit for performing the method for detecting nucleic acids in samples as described herein.
  • NA nucleic acid
  • dPCR digital polymerase chain reaction
  • CNVs gene copy number variations
  • SNPs single-nucleotide polymorphisms
  • titer plate-based digital PCR runs reactions in titer plates.
  • DNA molecules are diluted and partitioned into, e.g., 96-well titer plates, for independent PCR reactions. After amplification, all wells are probed and quantified by gene-specific sequences to identify wells with positive reactions.
  • Digital PCR as a PCR-based technology for nucleic acid detection, normally requires paired primers for amplification and probe(s) for detecting target nucleic acids, where samples are diluted and partitioned so that nucleic acid fragments therein are separated for independent reactions, each of which with very limited number of the target nucleic acid molecules.
  • the PCR reaction can be individually carried out within each partition and the signal in each partition is determined as negative or positive and therefore the exact amount of copies of a nucleic acid sequence in the original sample can then be determined by counting the number of positive partitions (sequence detected) versus negative partitions (sequence not detected) based on Poisson distribution.
  • the experimental procedure of digital PCR comprises a few steps: 1) dilution of target DNA molecules; 2) partition of well-separated target DNA fragments into discrete droplets/chambers, each of which also contains other required components for amplification and signal detection; 3) PCR amplification using“paired” gene-specific primers targeting“internal” regions of gene(s) of interest; 4) detection of fluorescent signals using fluorescent probe(s); 5) quantification of positive and negative reactions based on Poisson distribution; and 6) cross-sample comparison to determine the significance.
  • Cell-free nucleic acid samples are easy-accessible, noninvasive genetic materials with increasing popularity for the diagnosis of a number of diseases (Wagner, 2012). These genetic materials are released from all cells in the body, including normal cells, diseased cells and microbes.
  • cell-free nucleic acids may present in many types of body fluids including blood, saliva, urine, vaginal discharge, seminal fluid, lymph and sweat (Chiu and Yu, 2019; Nai et al., 2017; Wagner, 2012).
  • cell-free nucleic acid samples are frequently of low quantity, making it difficult to manipulate and thus easy to get lost during experimental process.
  • cell-free nucleic acids are also highly fragmented. These represent a serious problem especially for such precious materials.
  • the present invention provides an improved digital PCR for nucleic acid detection in a sample, called digital T-oligo primed PCR (digital TOP-PCR).
  • digital TOP-PCR digital TOP-PCR
  • the digital TOP-PCR of the present invention features use of a homogeneous adapter (HA) ligated to the termini of all nucleic acid fragments in the sample and a single-type primer (T/U oligo) that recognizes a complementary sequence in HA and acts as both forward and reverse primers to perform amplification.
  • HA homogeneous adapter
  • T/U oligo single-type primer
  • the digital TOP-PCR of the present invention allows amplification of all nucleic acid fragments in the sample such that subsequent detection of target nucleic acids can be achieved with increased sensitivity, especially reducing the false negative rate.
  • the present invention provides a method for analyzing nucleic acids in a sample, said sample containing one or more linear, double stranded nucleic acid fragment(s) (NA fragment(s)), said method comprising the steps of:
  • T 3’-thymine
  • U 3'-uracil
  • T nucleotide overhang double- stranded homogenous adapter which comprises a P oligo strand carrying a 5'-phosphate for ligation to the 3'-A overhang NA fragment(s) and a T/U oligo strand carrying a 3'-T or 3'-U without a 5 '-phosphate, wherein the T/U oligo strand is complimentary to the P oligo strand except at the 3'-T or 3'-U of the T/U oligo strand;
  • step (g) includes determining a droplet/fraction to be positive or negative based on the intensity of a fluorescent signal and later to calculate the total number (counts) of droplets/fractions with a positive signal.
  • step (e) more than 50% of the partitions contain no more than one copy of the adapter-ligated NA fragment(s).
  • each partition contains at least one copy of the adapter-ligated NA fragment(s).
  • the NA fragment(s) comprises a nucleic acid sequence that is indicative of a healthy /diseased state of the subject.
  • the homogenous adapter of step (b) does not self-ligate.
  • the homogenous adapter of step (b) has 3'-T or 3’U overhang in its T/U oligo strand and 3'-non-A overhang in its P oligo strand.
  • the homogenous adapter of step (b) has one end which is 3'-T overhang and the other end which is a blunt end.
  • the sample is obtained from a body fluid, including, but not limited to, blood, urine, saliva, tears, sweat, breast milk, nasal secretions, amniotic fluid, semen, and vaginal fluid.
  • the NA fragment(s) in the sample are cell-free DNAs (cfDNAs).
  • step (a) prior to step (a) the method as descried herein further comprises performing an end-repair reaction to the NA fragments.
  • the PCR of step (f) is performed by oil emulsion or droplet PCR, or well-based PCR.
  • step (g) is performed by flow cytometry using fluorescent probes.
  • the method of the present invention comprise the steps of (a) to (g) and optional step (a)': if the NA fragment(s) include liner, single-stranded RNAs, subjecting the sample prior to step (a) to a reverse transcription-PCR (RT-PCR) to convert the RNAs to linear, double-stranded complementary DNA (cDNA).
  • RT-PCR reverse transcription-PCR
  • the present invention also provides a kit for performing the method for detecting nucleic acid fragment(s) in samples as described herein.
  • the kit comprises
  • adapter ligation reagents comprising a homogenous adapter, a ligation buffer and a ligase
  • the homogenous adapter comprises a P oligo strand carrying a 5'- phosphate and a T/U oligo strand carrying a 3'-T or 3-U without a 5'-phosphate, wherein the T/U oligo strand is complimentary to the P oligo strand except at the 3'-T or 3'-U of the T/U oligo strand
  • the homogenous adapter is capable of ligating to the nucleic acid fragment(s) at both ends, wherein the nucleic acid fragment(s) has a 3'- A overhang;
  • PCR reagents comprising a single-type primer (the sole primer) having the nucleic acid sequence of the T/U oligo strand, dNTPs, a PCR buffer, and a DNA polymerase;
  • detection reagents comprising one or more detectable probes having
  • the kit further comprises instructions for use, wherein the instructions for use comprise instructions for performing the method comprising steps (a) to (g) as described herein.
  • Fig. 1 shows that the procedure of the method of the present invention.
  • P oligo 5’-GTCGGAGTCTgcgc-3’ (SEQ ID NO: 24).
  • T-oligo 5’-AGACTCCGAC(T)-3’ (SEQ ID NO: 23).
  • FIG. 2 shows the differences between the method of the present invention and the conventional PCR-based detection method.
  • a cfDNA sample contains a pool of cfDNA fragments with random breakages from genomic origins.
  • conventional PCR- based detection method only cfDNA fragment "g" covering both the first given primer binding site and the second given primer binding site can be amplified and detected; as a result, the sensitivity of detection is limited and particularly when the nucleic acid content is low, the sensitivity can be even worse.
  • all cfDNA fragments can be evenly and universally amplified and besides after such amplification not only fragment "g" but also other fragments "a” to "f ' (with only one primer binding site or even without any of the primer binding sites), all originating from the same target pathogenic nucleic acid (the same genomic origin), can be detected, for example, using one or more probe(s) with detectable label(s) capable of specifically hybridizing with any region within the target pathogenic nucleic acid; as a result, the sensitivity of detection is increased and false negatives can be minimized since the amplification before detection is equally (non-specifically) applicable to all nucleic acid fragments and thus the relative amount of each of the amplified nucleic acid fragments can represent to that present in the original sample.
  • Fig. 3 shows the counts of positive droplets produced from conventional ddPCR and ddTOP-PCR methods using samples containing variable amounts of partial and full templates.
  • Lanes 1 and 2 (A05 and A06 controls) were generated from 5’ primer binding site-deleted NAGK gene sequence using T/U oligo primer and N-myc gene-specific primers, respectively, for amplification, while the rest (lanes 3 - 16, or B05 - H06) are generated from mixed templates containing variable amounts (100% - 0%) of partial (labeled as ⁇ ’, or 5’ primer binding site-deleted) and full (labeled as‘F’, or with both primer binding sites) templates. All samples used the same N-myc probe for detection.
  • Counts from ddTOP-PCR are labeled with‘05’ behind letter, while their corresponding counterparts of ddPCR are labeled side-by-side with‘06’. Digits in the figure represent counts (copies/microliter). A total of 20 microliter for each sample was used for counting by QX200 ddPCR machine. Chl, channel 1 as defined by QX200. [0031] Fig. 4 shows the comparison of fluorescent signal intensity between ddPCR and ddTOP-PCR. Same as Fig.
  • lanes 1 and 2 were generated from 5’ primer binding site-deleted NAGK template using T/U oligo primer and N-my c primers, respectively, for amplification, while the rest (lanes 3 - 16, or B05 - H06) are generated from mixed templates containing variable amounts (100% - 0%) of partial (labeled as ⁇ ’, 5’ primer binding site-deleted) and full (labeled as‘F’, with both primer binding sites) templates. All samples used the same N-myc oligo probe for detection. Notice that, droplets shown as black dots were not counted as positives, because their intensities were below default threshold.
  • Fig. 5 shows the sequences as set forth in Table 1.
  • T oligo 5’-AGC GCT AGA CTC CGA CT-3’ (SEQ ID NO: 1).
  • P oligo, 5’-GT CGG AGT CTA GCG CT-3’ SEQ ID NO: 2.
  • the articles “a” and “an” refer to one or more than one (i.e., at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • “around”,“about” or“approximately” can generally mean within 20 percent, particularly within 10 percent, and more particularly within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term“around”,“about” or“approximately” can be inferred if not expressly indicated.
  • polynucleotide or“nucleic acid” refers to a polymer composed of nucleotide units.
  • Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well as nucleic acid analogs including those which have non-naturally occurring nucleotides.
  • Polynucleotides can be synthesized, for example, using an automated DNA synthesizer.
  • the term“nucleic acid” typically refers to large polynucleotides.
  • oligonucleotide refers to a relatively short nucleic acid fragment, typically less than or equal to 150 nucleotides long e.g., between 5 and 150. Oligonucleotides can be designed and synthesized as needed. In the case of a primer, it is typically between 5 and 50 nucleotides, particularly between 8 and 30 nucleotides in length. In the case of a probe, it is typically between 10 and 100 nucleotides, particularly between 15 and 30 nucleotides in length.
  • the term “complementary” refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides.
  • the two molecules can be described as complementary, and furthermore the contact surface characteristics are complementary to each other.
  • a first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide.
  • the polynucleotide whose sequence 5'- TATAC-3' is complementary to a polynucleotide whose sequence is 5'-GTATA-3'.”
  • target nucleic acids can refer to particular nucleic acids of interest being detected in a sample.
  • Target nucleic acids include but are not limited to DNA such as genomic DNA, mitochondrial DNA, cDNA and the like, and RNA such as mRNA, miRNA, and the like.
  • Target nucleic acids may derive from any sources including naturally occurring sources or synthetic sources.
  • target nucleic acids may be from animal or pathogen sources including, without limitation, mammals such as humans, and pathogens such as bacteria, viruses and fungi.
  • Target nucleic acids can be obtained from any body fluids or tissues (e.g., blood, urine, skin, hair, stool, and mucus), or an environmental sample (e.g., a water sample or a food sample).
  • target nucleic acids can be a collection of nucleic acid molecules of the same origin (e.g., from the same gene of normal or diseased subject or pathogens) but in various length. For example, numerous segments of the gene encoding for hepatitis B surface antigen (HBsAg) may be present in a test sample as "target" nucleic acids fragments of various length. Since each of the target nucleic acid molecules contains at least a portion of the HBsAg gene, probes or primers having sequences corresponding (or complementary) to various locations within the HbsAg gene can be used for detection of the target nucleic acid fragments. For another example, target nucleic acids may be those containing genetic mutations (e.g., a single nucleotide polymorphism (SNP) indicative of a disease such as cancer).
  • SNP single nucleotide polymorphism
  • primer refers to oligonucleotides that can be used in an amplification method, such as a polymerase chain reaction (PCR), to amplify a target nucleotide sequence.
  • PCR polymerase chain reaction
  • at least one pair of primers including one forward primer and one reverse primer are required to carry out the amplification.
  • a forward primer is an oligonucleotide that can hybridize to the 3' end of the (-) strand and can thus initiate the polymerization of anew (+) strand under the reaction condition
  • a reverse primer is an oligonucleotide that can hybridize to the 3' end of the (+) strand under the reaction condition and can thus initiate the polymerization of a new (-) strand under the reaction condition.
  • a forward primer may have the same sequence as the 5' end of the (+) strand
  • a reverse primer may have the same sequence as the 5' end of the (-) strand.
  • a forward primer and a reverse primer useful for amplification of a target nucleic acid sequence are different from each other in sequence.
  • a single primer refers to only one type of primer, all of which have the same sequence, instead of a pair of primers having distinct sequences, one being a forward primer and the other being a reverse primer.
  • hybridization shall include any process by which a strand of nucleic acid joins with a complementary strand through base pairing.
  • Relevant methods are well known in the art and described in, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 nd ed., Cold Spring Harbor Laboratory Press (1989), and Frederick M.A. et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (2001).
  • stringent conditions are selected to be about 5 to 30°C lower than the thermal melting point (T m ) for the specified sequence at a defined ionic strength and pH.
  • stringent conditions are selected to be about 5 to l5°C lower than the T m for the specified sequence at a defined ionic strength and pH.
  • stringent hybridization conditions will be those in which the salt concentration is less than about 1.0 M sodium (or other salts) ion, typically about 0.01 to about 1 M sodium ion concentration at about pH 7.0 to about pH 8.3 and the temperature is at least about 25°C for short probes (e.g., 10 to 50 nucleotides) and at least about 55°C for long probes (e.g., greater than 50 nucleotides).
  • An exemplary non-stringent or low stringency condition for a long probe would comprise a buffer of 20 mM Tris, pH 8.5, 50 mM KC1, and 2 mM MgCl 2 , and a reaction temperature of 25°C.
  • an "overhang” refers to a stretch of a single unpaired nucleotide or a longer unpaired nucleotides at the end of a linear, double-stranded nucleic acid molecule.
  • the unpaired nucleotide(s) can be in either 3’ or 5’ end, creating either 3' or 5' overhangs, respectively.
  • A“3'-A overhang” means the unpaired nucleotide(s) is/are present in 3'-end and it is composed of one or more adenine (A) nucleotides.
  • A“3'- non-A overhang” means the unpaired nucleotide(s) is/are present in 3'-end and it does not include any adenine (A) nucleotide.
  • a “3-T overhang” means the unpaired nucleotide(s) is/are present in 3'-end and it is composed of one or more thymine (T) nucleotide.
  • a "single,” “homogenous” or “universal” primer means only one type of primer with the same sequence is present, instead of a pair of primers, in the PCR reaction.
  • the term“heterogeneous primers” means at least one paired primers each member having different sequences from each other are present in the PCR reaction.
  • the term "adapter” refers to an oligonucleotide that can be ligated to the ends of a double-stranded nucleic acid molecule.
  • An adapter may be 10 to 50 bases in length, preferably 10 to 30 based in length, more preferably 10 to 20 based in length. Lower than 10 nucleotide in length may decrease specificity for annealing. Higher than 20 nucleotides in length may not be cost-effective.
  • the term a "homogeneous" adapter means one single type of adapter for ligating to both ends of a double-stranded nucleic acid molecule.
  • a heterogeneous adapter means at least two types of adapters that have different nucleotide sequences from each other, one for ligating to 5' end and the other for ligating to 3 'end of a double-stranded nucleic acid molecule.
  • T-oligo- primed polymerase chain reaction TOP-PCR
  • a homogenous adapter composed of a P-oligo and a T-oligo and ligated to both ends of all the nucleic acid fragments and then applying the T-oligo as a single primer to perform the amplification of all the nucleic acid fragments in the sample without discrimination.
  • the method of the present invention is performed for nucleic acid detection in a digital manner by using TOP- PCR for amplification so that all the nucleic acids in the reaction are amplified in equal proportion and target nucleic acids can be detected by one or multiple sequence-specific probes with increased sensitivity.
  • the method of the present invention shows at least about 14% increase in sensitivity compared to conventional digital PCR using a paired PCR primer for amplification.
  • Fig. 1 is a diagram showing the procedures of the method of the present invention.
  • DNA samples can be obtained from any sample containing particular nucleic acids of interest to be detected, for example, bodily fluid or tissue e.g. blood, urine, skin, hair, stool, and mucus, or an environmental sample e.g. a water sample or a food sample.
  • the samples can be treated to isolate and purify DNAs therefrom by routine procedures such as phenol-chloroform extraction or Qiagen kit.
  • the method of the present invention can be used for both DNA and RNA targets.
  • DNA polymerase can be used directly for amplification.
  • RNA samples a reverse transcription step with reverse transcriptase will need to be first performed.
  • the DNA fragments in sample are end-repaired, tailed an "A" to every 3’ end, to provide 3'A-overhang DNA fragments.
  • Conventional methods or kits are available to perform the end repair and A-tailing step such as NEBNext® Ultra End Repair/dA- Tailing Module (NEB, E7442S/L).
  • a homogenous adapter is designed for use in TOP-PCR amplification.
  • one strand is called T/U oligo, having an extra thymine or uracil nucleotide (T/U) at the 3'-end; and the other strand is called P oligo, having a phosphate group at the 5'-end and its 3'-end nucleotide has no extra T or U.
  • the adapter may be a blunt-sticky (i.e., one end is blunt and the other end is sticky) or double sticky (i.e., both ends are sticky) adapter.
  • the P oligo is one base shorter and is complementary to the T/U oligo except at the 3'-end T/U of the T/U oligo. In some embodiments, for a“double-sticky” adapter, the P oligo is longer than the T/U oligo.
  • a homogenous adapter as used herein requires (i) T/U oligo has an extra 3’- T/U (i.e.,‘ or‘IT overhang at the 3’ end) and has no 5’-phosphate; (ii) P oligo needs a 5’-phosphate; and (iii) T/U oligo is complimentary to P oligo except at the 3’- T/U overhang of T/U oligo.
  • T/U oligo and P oligo may vary in length and sequence.
  • P oligo sequence is 5’-GTCGGAGTCTgcgc-3’ (SEQ ID NO:24)
  • T/U-oligo sequence is 5’-AGACTCCGAC(T)-3’ (SEQ ID NO:23).
  • P oligo sequence is 5’- GT CGG AGT CTA GCG CT -3’ (SEQ ID NO: 2)
  • T/U-oligo sequence is 5’- AGC GCT AGA CTC CGA CT -3’ (SEQ ID NO: 1).
  • “3’-U”, instead of 3 -T . can be used, so that the double-stranded half adapter (HA) can be completely trimmed off after amplification by using“user enzyme” (Uracil-Specific Excision Reagent, by NEB).
  • the homogenous adapter is ligated to both termini of the 3'A-overhang DNA fragments after the end repair and A-tailing step, to produce adapter-ligated DNA fragments, wherein the 3'-T/U of the T/U oligo of the adapter is complementary to the 3- ⁇ overhang of the 3'-A overhang DNA fragments.
  • the ligation can be conducted in a proper ligation mixture including the adapter, the 3 '-A overhang DNA fragments, ligase and ligation buffer, under a proper condition e.g. about 25 °C overnight in a thermocycler.
  • the ligation mixture can be directly subjected to PCR amplification with or without DNA purification.
  • PCR reagents typically include primers, nucleotides, polymerase, and buffers.
  • Detection reagents typically include one or more detectable probe(s). These input reagents may be provided as individual reagents to be added separately into the sample, or some or all of the reagents may be provided as reagent mixes to be added into the sample in a premixed form.
  • PCR reagents typically include a buffer which is selected to facilitate the amplification reaction. Magnesium ions e.g. MgCh are usefully included in the buffer.
  • PCR reagents also include nucleotides.
  • the four dNTPs (dATP, dCTP, dGTP and dTTP) are typically provided in equimolar concentrations.
  • dNTPs dATP, dCTP, dGTP and dTTP
  • a variety of PCR polymerases may be used in the same to perform amplification. Suitable polymerases will often have optimal activity at about 75° C. and the ability to retain that activity after prolonged incubation, e.g., at temperatures greater than 95° C.
  • Useful polymerases may include, for example, Taq DNA polymerases, such as AmpliTaq®, AmpliTaq Gold®, the Stoffel fragment of AmpliTaq®, and others.
  • the PCR reagents include a single-type primer having the nucleic acid sequence of the T/U oligo strand as described herein as both forward and reverse primers for amplification.
  • Detection reagents including a probe with specificity for a target nucleic acid can be added to the sample and a detectable signal e.g. a fluorescence signal caused by degradation of the probe can be detected.
  • the amplification/detecti on-ready sample is fractioned into multiple partitions to an extent that each containing limited copies of the adapter-ligated NA fragments. Specifically, most partitions may contain no copies, others only one copy, still others could contain two copies, three copies, and even higher numbers of copies. Copies per partition may be adjusted as needed.
  • the fractionation is carried out to an extent that more than 50% of the partitions containing no more than one copy of the adapter-ligated NA fragment(s). In some embodiments, the fractionation is carried out to an extent that each partition contains at least one copy of the adapter- ligated NA fragment(s) e.g. 1-5 copies in each partition.
  • the fractionation can be carried out in emulsion droplets or in a multiple-well as known in the art, for example, as described in Lodrini et al, 2017 and U.S. Patent Application Publication No. 2009/0053719 and 20150099644, the entire content of which is incorporated herein by reference.
  • the sample is divided into a plurality of small reactions in oil droplets through a water oil emulsion technique.
  • the oil droplets are made using a droplet generator. Typically, approximately 20,000 oil droplets are formed from each 20 m ⁇ sample.
  • a free T/U oligo is a single primer having the nucleic acid sequence of the T/U oligo strand as described herein.
  • a free T/U oligo refers to a T/U oligo that is not formed in an adapter with its complementary P oligo. In this way, all the DNA fragments ligated with the adapter at both end are amplified in equal proportion.
  • Detection of the target DNAs can be performed by a number of methods known in the art such as flow cytometry using fluorescent probes.
  • a probe having a detectable label such as a fluorophore (e.g. FAM, 6-fluorescein amidite) is used for detection.
  • a fluorescent probe has complementary sequences which specifically hybridize with a target nucleic acid fragment where the fluorophore is released from the probe during amplification of the target nucleic acid fragment by PCR (generating a positive signal, indicating sequence detected) while the fluorophore is not released from the probe if target nucleic acid fragment is not present or no target nucleic acid fragment is amplified (generating a negative signal, indicating sequence not detected).
  • a detectable probe is present in the PCR mixture.
  • fragment "g” (covering two primer binding sites) but also other fragments "a” to "f ' (with only one primer binding site or even without any of the primer binding sites), all originating from the same target pathogenic nucleic acid, can be detected, for example, using shotgun probe(s) with detectable label(s) capable of specifically hybridizing with any region within the target DNAs, such that sensitivity of detection is increased and false negatives can be minimal.
  • the number of positive partitions (sequence detected) versus negative partitions (sequence not detected) is counted to determine the estimated amount of the target nucleic acid fragment in the sample.
  • the quantification can be carried out according to a method known in the art, for example, as described in Lodrini et al, 2017.
  • droplets after PCR amplification can be measured in the QX200 ddPCR Droplet Reader and target copy number is analyzed using QuantaSoft analysis software.
  • kits for performing the method for detecting nucleic acid fragment(s) in samples as described herein comprises
  • adapter ligation reagents comprising a homogenous adapter, a ligation buffer and a ligase
  • the homogenous adapter comprises a P oligo strand carrying a 5'- phosphate and a T/U oligo strand carrying a 3'-T or 3-U without a 5'-phosphate, wherein the T/U oligo strand is complimentary to the P oligo strand except at the 3'-T or 3'-U of the T/U oligo strand
  • the homogenous adapter is capable of ligating to the nucleic acid fragment(s) at both ends, wherein the nucleic acid fragment(s) has a 3'- A overhang;
  • PCR reagents comprising a single-type primer having the nucleic acid sequence of the T/U oligo strand, dNTPs (dATP, dCTP, dGTP and dTTP), a PCR buffer, and a DNA polymerase; and
  • detection reagents comprising one or more detectable probes having complementary sequences specifically hybridizing with the nucleic acid fragment(s).
  • the kit further comprises instructions for use.
  • the instructions for use comprise instructions for performing the method of the present invention comprising steps (a) to (g).
  • the method of the present invention is useful in diagnosis or prognosis, especially in cfDNA-based detection. Detection of body fluid samples containing cfDNAs has been described a non-invasive approach useful for diagnostics of genetic defects, infectious origins and diseases, especially valuable in early detection, and also prognosis at least because cfDNA remains available even if the diseased tissues e.g. tumors are removed.
  • conventional PCR including qPCR or dPCR, designed as template-dependent requiring at least one one pair of primers, is not suitable for cfDNA detection since the cfDNA as the templates are usually not in good quality and quantity such that sensitivity has limitation and bias may happen if PCR cycles increase.
  • the method of the present invention by using a homogenous adapter composed of a P oligo and a T/U oligo ligating to the DNAs and the T/U oligo as a single primer, is able to amplify all the DNAs in a sample with any initial quantity, in equal proportion, and detection of target DNAs with specific probes can be carried out to increase the sensitivity without substantial bias (false negative).
  • dPCR digital PCR
  • CNVs copy number variations
  • ddTOP-PCR droplet digital T/U oligo-primed polymerase chain reaction
  • ddTOP-PCR showed -14% increase in sensitivity, compared to conventional ddPCR, although with compromised signal intensity.
  • N-myc amplicon (157 bp) was cloned/amplified from the genomic DNA of Be2C cell line using forward primer 5’- AAG GGG TGC TCT CCA ATT CT-3’ (SEQ ID NO: 13) and reverse primer 5 -CGG TTT AGC CAC CAA CTT TC-3’ (SEQ ID NO: 14).
  • NAGK amplicon (172 bp) was cloned/amplified from the genomic DNA of Be2C cell line using forward primer 5’- CCC CTT TCC CGC TAT ATC TT-3’ (SEQ ID NO: 15) and reverse primer 5’-ATG CAG GGT TTG ATG GGATA-3’ (SEQ ID NO: 16).
  • Polymerase chain reaction (PCR) using Q5 High-Fidelity 2X Master Mix (NEB, MA, US) was carried out to amplify the target amplicon. PCR reaction was performed in a mixture (50 pl) containing the respective primer set, Be2C genomic DNA (10 ng), l x Q5 High-Fidelity Master Mix.
  • the mixture was then incubated at 98°C for 1 min followed by 30 cycles of 98°C for 20 s, 60°C for 30 s and 72°C for 10 s. The mixture was later incubated at 72°C for 2 min for final elongation.
  • the PCR products were then analysed and amplicons with the expected size were extracted from a 2% agarose gel prior gel DNA extraction using QIAquick Gel Extraction Kit (Qiagen, NW, GE) following the instruction manual.
  • Half-adapter was first prepared by annealing l6-mer P oligo (5’- pGTCGGAGTCTAGCGCT-3C6-3’) (SEQ ID NO: 2) and l7-mer T/U oligo (5’- AmC6-AGCGCTAGACTCCGACT-3’) (SEQ ID NO: 1) at 1: 1 molar ratio by incubating at 95°C for 5 min followed by gradually reduction of temperature to 4°C using a thermocycler.
  • the mixture was then incubated at 98°C for 1 min preceded 30 cycles of 98°C for 20 sec, 57°C for 30 sec and 72°C for 1 min for sheared gDNA while 10 sec for the standard templates. The mixture was later incubated at 72°C for 5 min for final elongation.
  • the TOP-PCR amplified N-myc and NAGK namely HA-N-myc-HA and HA-NAGK-HA respectively were purified using QIAquick PCR Purification Kit (Qiagen, NW, GE) following the instruction manual and quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, MA, US).
  • the ddPCR reaction (20 m ⁇ ) consists T/U oligo primer (8 mM), Mpbl+2 (0.25 mM), Npb2 (0.25 mM), DNA template (4.0 m ⁇ ), 1 c ddPCRTM Supermix for Probes (No dUTP) (Bio- Rad, CA, US) prior droplet generation.
  • the ddPCR droplet mixtures were prepared and ddPCR reaction were performed as aforementioned.
  • the ddPCR reaction (20 m ⁇ ) consists T/U oligo primer (8 mM), Mpbl+2 (0.25 mM), Npb2 (0.25 mM), gDNA-HA template (100 ng to 100 p g), U ddPCRTM Supermix for Probes (No dUTP) (Bio-Rad, CA, US) prior droplet generation.
  • the ddPCR droplet mixtures were prepared and ddPCR reaction were performed as aforementioned.
  • HA-N myc-HA and HA-NAGK-HA constructs was then cloned using HE Swift Cloning Kit (Toolbiotech, TW), followed by transformation into DH5a competent cells and plating onto ampicillin LB agar plate. Bacteria colonies were screened and sequenced via Sanger sequencing to verify the sequences after plasmid extraction using QIAprep Spin Miniprep Kit (Qiagen, NW, GE).
  • the standard templates for ddTOP-PCR were produced by amplifying the recombinant plasmid harbouring the correct HA-N myc-HA and HA-NAGK-HA sequences in a 100 m ⁇ PCR reaction containing the pHE-F primer (5’-CGA CTC ACT ATA GGG AGA GCG GC-3’; SEQ ID NO: 17, 0.5 mM), pHE-R primer (5’-AA GAA CAT CGA TTT TCC ATG GCA G-3’; SEQ ID NO: 18, 0.5 mM), DNA (1 ng), l x Q5 High-Fidelity Master Mix.
  • the mixture was then incubated at 98°C for 1 min followed by 30 cycles of 98°C for 20 s, 64°C for 30 s and 72°C for 10 sec. The mixture was later incubated at 72°C for 2 min for final elongation.
  • the PCR amplicon namely pHE-HA- N myc-HA and pHE-HA-NAGK-HA with size of 309 bp and 325 bp respectively were purified using QIAquick PCR Purification Kit (Qiagen, NW, GE) and quantified.
  • the 5’ primer binding site-deleted construct of N-myc was amplifying with forward primer (5’-AGC GCT AGA CTC CGA CTT CAC TAAAGT TCC TTC CAC CCT CTC CTG GGG AG-3’) (SEQ ID NO: 19) and reverse primer (5’-AGC GCT AGA CTC CGA CTTAGC CAC CAA CTT TCT CCA ATT TTA TTC CTC AG-3’) (SEQ ID NO: 20) by Q5 High-Fidelity Master Mix.
  • forward primer 5’-AGC GCT AGA CTC CGA CTT CAC TAAAGT TCC TTC CAC CCT CTC CTG GGG AG-3’
  • reverse primer 5’-AGC GCT AGA CTC CGA CTTAGC CAC CAA CTT TCT CCA ATT TTA TTC CTC AG-3’
  • the 5’ primer binding site-deleted construct of NAGK was amplifying with forward primer (5’-AGC GCT AGA CTC CGA CTG TGT TGC CCG AGA TTG ACC CGG TGA GTT GAG GT-3’) (SEQ ID NO: 21) and reverse primer (5’-AGC GCT AGA CTC CGA CTA TGC AGG GTT TGA TGG GAT AGT CCC ATC-3’) (SEQ ID NO: 22) by Q5 High-Fidelity Master Mix.
  • forward primer 5’-AGC GCT AGA CTC CGA CTG TGT TGC CCG AGA TTG ACC CGG TGA GTT GAG GT-3’
  • reverse primer 5’-AGC GCT AGA CTC CGA CTA TGC AGG GTT TGA TGG GAT AGT CCC ATC-3’
  • the PCR amplicon namely HA-N myc-F del -HA and HA-NAGK-F del -HA with size of 160 bp and 146 bp respectively were purified using QIAquick PCR Purification Kit (Qiagen, NW, GE) and quantified.
  • Paired primer sequences for the ddPCR were retrieved from Lodrini et al., with slight modification on the sequence (Lodrini et al, 2017).
  • the primers and probes were synthesized by Integrated DNA Technology (IDT).
  • IDT Integrated DNA Technology
  • forward primer (5’-GTG CTC TCC AAT TCT CGC CT-3’) (SEQ ID NO: 3) and reverse primer (5’-GAT GGC CTA GAG GAG GGC T-3’) (SEQ ID NO: 4) are employed.
  • probe Mpbl FAM-N-myc probe
  • Mpbl+l FAM-N-myc Probe +lnt
  • probe Mpbl+2 FAM-N-myc Probe +2nt
  • a total of 20 m ⁇ PCR reaction comprises primer(s) (paired primers for ddPCR control or aT/U oligo primer for ddTOP-PCR), 0.25 mM probe, DNA template (2.0 m ⁇ ), 1 x ddPCR Supermix for probes (No dUTP) (Bio-Rad). Droplets were generated by mixing the prepared PCR reaction mixture (20 m ⁇ ) with 70 m ⁇ of droplet digital PCR oils (Bio-Rad).
  • Samples containing only NAGK or N-myc sequence provides an easily verifiable system to improve conditions, under which experimental conditions (e.g., preparation of samples, concentrations of PCR ingredients and reaction conditions) can be adjusted based on experimental outcome.
  • experimental conditions e.g., preparation of samples, concentrations of PCR ingredients and reaction conditions
  • Results indicate that ddPCR was able to detect about 48.6% of the templates, while ddTOP-PCR was able to detect about 58.5% - 62.8% of the templates, indicating a 10% - 15% increase in sensitivity from ddPCR to ddTOP-PCR. Notice that, although accuracy is influenced by bias/variations resulted from quantification device (e.g., Qubit), personal skill, QX200 machine itself, and others, the general trend for each method possesses certain degree of reliability.
  • quantification device e.g., Qubit
  • color intensities of droplets produced from ddTOP-PCR can be either higher or low and are more scattered than that produced from ddPCR.
  • ddPCR was unable to detect 5’ primer binding site-deleted fragment, while ddTOP-PCR detects it with high efficiency.
  • the first two lanes in the figure indicate low levels of false positives for NAGK templates. Previous observations indicate that null background has clean count (0) for both ddTOP-PCR and ddPCR method (data not shown).
  • ddTOP-PCR Comparing to conventional ddPCR, ddTOP-PCR has a number of advantages: 1) Digital PCR is not suitable for cfDNA analysis, because, as a template- dependent method, regular PCR requires both primer-binding sites to co-present in the same fragment. On the other hand, as an adapter-dependent PCR method, ddTOP-PCR does not have such constrain and thus is able to detect partial fragments; 2) Prior to ddTOP-PCR experiment, TOP-PCR alone can be employed to preserve low-quantity samples, while conventional PCR cannot.
  • ddTOP-PCR may be suitable for early detection of cancer and possibly other diseases as well, while conventional ddPCR cannot.
  • Lodrini M., Sprussel, A., Astrahantseff, K., Tiburtius, D., Konschak, R., Lode, H.N., Fischer, M., Keilholz, U., Eggert, A., and Deubzer, H.E. (2017).
  • T Obgo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids. Sci Rep 7, 40767.

Abstract

La présente invention concerne un procédé pour la détection de molécules d'acide nucléique (AN) dans des échantillons. Plus particulièrement, la présente invention concerne un procédé amélioré basé sur la PCR numérique pour détecter une ou plusieurs séquences d'acide nucléique spécifiques. La présente invention est avantageuse pour des applications de recherche et de diagnostic avec une sensibilité et une précision accrues. La présente invention concerne également un kit pour mettre en œuvre le procédé pour analyser des acides nucléiques dans des échantillons tels que décrits dans la description.
PCT/US2019/055342 2018-10-09 2019-10-09 Procédé de réaction en chaîne par polymérase numérique permettant la détection d'acides nucléiques dans des échantillons WO2020076914A1 (fr)

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EP19871402.4A EP3864658A4 (fr) 2018-10-09 2019-10-09 Procédé de réaction en chaîne par polymérase numérique permettant la détection d'acides nucléiques dans des échantillons
US17/283,809 US20210340611A1 (en) 2018-10-09 2019-10-09 Digital polymerase chain reaction method for detecting nucleic acids in samples

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